The present invention relates generally to delivery of compounds with a net negative charge, especially oligonucleotides, to specific cell types, and is in particular a means of using positively-charged porphyrins and other macrocyclic compounds with positive charges that can stack along oligonucleic acid backbones to stabilize and promote uptake into cells of the oligonucleotides or other negatively charged compounds. The present invention is also a method for treating viral diseases, especially hepatitis B and C.
Targeted drug delivery improves the therapeutic index of numerous drugs, reduces potential drug cost and may increase tissue half life. Although drugs can be encapsulated in tablets or capsules for oral delivery, encapsulation into more sophisticated vehicles is required for targeted delivery and for delivery of molecules such as therapeutic oligonucleotides and gene therapy reagents, which are extremely sensitive to the presence of nucleases in the body.
Many different systems have been proposed for targeted drug delivery. The most commonly used method has been to covalently attach antibodies to the surface of microparticulate carriers.
Delivery of short nucleic acids to the liver is a crucial step in their use as genetic therapeutics in hepatic illness. In hepatitis caused by the human hepatitis viruses, the hepatocytes are the sites of intracellular viral replication and are thus the target cells for antiviral therapies. Receptor-mediated uptake of oligonucleotides into hepatocytes has been used as a strategy for the specific delivery, as described by Wu and Wu (1986) Receptor-mediated in vitro gene transformation by a soluble DNA carrier system J. Biol. Chem. 262, 4429-4432. Receptors such as the asialoglycoprotein receptor and the heme receptor (Galbraith, R. A. (1990) Heme binding to HepG2 human hepatoma cells. J. Hepatol., 10, 305-310) have been successfully targeted. Immunoliposomes, i.e., liposomes bearing antibodies have also been used to direct drug delivery. However, the coupling of polypeptide ligands to the surface of liposomes presents a number of problems, mainly due to the fact that such ligands contain multiple reactive groups. Carbodiimide-mediated peptide bond formation between complex ligand molecules such as carbohydrates or polypeptides and reactive groups on the outer surface of liposomes can result in considerable intramolecular coupling and intermolecular coupling between ligand molecules, in addition to the desired intermolecular coupling between liposome and ligand. Complex ligands are also more likely to be immunogenic and could therefore evoke an immune reaction resulting in rapid clearance by the immune system of the body.
It is therefore an object of the present invention to provide a means for stabilizing and delivering nucleic acids.
It is a further object of the present invention to provide compositions for efficient, simple and reliable delivery to specific cell types, especially of nucleic acid-type molecules, such as external guide sequences for RNase P, antisense oligonucleotides and ribozymes.
It is another object of the present invention to provide methods and compositions for treating hepatotrophic viruses; especially human hepatitis B and C viruses.
Efficient methods and compositions are provided for delivery of effective concentrations of compounds, including nucleic acid molecules and oligonucleotides such as ribozymes, external guide sequences for RNase P, and antisense oligonucleotides, proteins, peptides carbohydrate, and other synthetic organic and inorganic molecules having biological activity or useful as a diagnostic, or combinations thereof, to cells, especially hepatocytes and tumor cells, which preferentially bind prophyrins or phthalocyanins (referred to jointly herein as xe2x80x9cporphyrinsxe2x80x9d unless otherwise stated) or other macrocylic compounds. The system is extremely simple, since the two principle components are a porphyrin having a net overall positive charge, as defined in more detail below, and the compound to be delivered, wherein the compound has a net overall negative charge. The porphyrin binds the compound to be delivered and selectively targets the compound to cells preferentially binding the porphyrin.
As demonstrated by the examples, in a preferred embodiment the compound is an oligonucleotide which binds to the porphyrin in a stoichiometric ratio, and greatly enhances uptake by cells. The examples demonstrate delivery to hepatocytes, lack of side effects in animals, inhibition of viral replication due to both the oligonucleotide and porphyrins, with a significant enhancement of the anti-viral activity due to the combination of oligonucleotide and porphyrin. The combination has utility in inhibition of viral replication in cells, as well as other therapeutic applications, as well as in research and diagnostic applications.
Also disclosed is the use of the porphyrins alone as anti-human hepatitis B agents, having anti-viral activity in the absence of the anti-viral oligonucleotide.